As the biotechnology, pharmaceutical and chemical industries grow, their laboratory research and development proportionately expands. Thus, research precision has become increasingly important, often distinguishing the efforts of one company or group from another. Thus, research apparatus and techniques, once thought to represent the industry standard, are continually being replaced by better, more efficient and more accurate apparatus and techniques.
In particular, the use of pipetting assemblies has rapidly increased in volume and simultaneously has increasingly required more accuracy, as minute differences often dictate success or failure. Pipette assemblies have traditionally included two basic components, namely, the pipetter and the hollow pipette tip. The pipetter is a device for creating a pressure differential inside the pipette tip. When a negative pressure or vacuum is present in the tip, liquid is drawn into the tip. When a positive pressure is created, or the vacuum is released, the liquid is forced from or gravitates from the tip.
Early pipette assemblies employed relatively simple pipetters and pipette tips which included volumetric marks or lines. The technician would merely draw fluid into the tip until it reached the reference line. The accuracy of this approach depended upon the technician's skill and the accuracy of calibration of the reference marks, neither one of which could be safely relied upon when a high degree of precision was required.
Subsequently, considerable effort has been directed toward providing adjustable mechanical and electromechanical pipetters which can be adjusted to a volume and then have that volume calibrated. Pipetter devices can be divided into two broad categories, namely, the "to-deliver" and the "to-contain" pipetters. A "to-deliver" pipetter overdraws the amount of fluid desired, but delivers, dispenses or discharges an only pre-determined or adjusted volume, which can be accurately calibrated. "To-contain" pipetters draw an adjusted volume of liquid and then dispense or discharge that entire volume. Regardless of the type of mechanical pipetter, they typically employ a piston and cylinder arrangement which, through air displacement, draws the desired fluid into the disposable pipette tip and delivers or dispenses the liquid from the tip at a later time.
FIG. 1 illustrates a typical mechanically adjustable pipetter device 10 which includes a piston and cylinder assembly (not shown). The piston and cylinder are generally of a known diameter and the stroke of the piston is coupled to a volume adjustment mechanism 16 having a vernier scale or indicator 12 shown proximate the upper end of pipetter 10. In pipette assembly 10, volume adjustment mechanism 16 also includes a displaceable button 14. Volume indicia 18 at indicator 12 allows the technician to set the proposed volume of fluid to be drawn or dispensed. By accounting for the differences in the specific gravities between the air and the desired fluid, as well as the viscosity difference, the drawn volume of liquid can be reasonably accurately set. Once the desired volume is set, button 14 may be depressed to displace air from the cylinder. Upon the return stroke of the piston, a corresponding volume of fluid is drawn into a reservoir or pipette tip 20.
Typical of adjustable pipetters are the pipetters sold under the trademarks EPPENDORF.RTM., PIPETMAN.RTM., FINNPIPETTE, EXCALIBUR.RTM., SOCOREX and OXFORD.RTM., to name a few. One problem which exists with these adjustable pipetters is that the volume indicated on the volume adjustment mechanism often is not an accurate determination of the actual amount of fluid withdrawn or dispensed. The pipette volume, generally, needs to be calibrated every time the volume is adjusted or set. Furthermore, even once fixed and calibrated, the volume should be recalibrated every week, although this is rare in actual practice. Thus, even adjustable pipetters having supposedly known or calibrated dispensing volumes will dispense volumes of liquid which vary from the nominal pre-set or adjusted volume.
Another problem associated with calibrated mechanical pipetters 10 is that various environmental factors, such as room temperature and atmospheric pressure, influence the accuracy of the fluid volume drawn or dispensed. Additionally, pipetter 10 often will be cradled in the research technician's hand, which may raise the temperature of the piston/cylinder arrangement so that thermal expansion causes inaccurate draws or deliveries.
A further problem experienced by adjustable mechanical pipetters is that the internal mechanisms can wear or fatigue. Excessive use may deteriorate the mechanical parts, for example, the spring used to urge the piston upward during the fluid draw. A worn or fatigued spring may not urge the piston to its full draw or may lead to inconsistencies on successive draws. Moreover, the O-ring assemblies may crack or leak causing the piston/cylinder arrangement to lose pressure. These above-mentioned mechanical breakdowns, in addition to the research technician's level of competence and/or fatigue, vary the fluid volume draws or deliveries, even with a "calibrated" adjustable mechanical pipetter.
Briefly, pipette tips 20 are usually provided by disposable plastic tips which are slidably and frictionally held on a tip mounting portion 22 located at the bottom end of pipetter 10. Pipette tips 20 may be hollow cylindrical or conical members which extend from a pipetter mounting end 24 to a liquid intake end 26.
In an attempt to alleviate some of the problems encountered in connection with adjustable pipetters, disposable pipette tips 20 having visual indicators or markings for estimating the amount of the drawn or dispensed fluids have been used with mechanical pipetters. As shown in FIG. 2, a disposable pipette tip 20' of the prior art is illustrated which can be slidably mounted to tip mounting portion 22 of pipetter assembly 10, shown in FIG. 1. This pipette tip is commercially available under the trademark REFERENCE TIP.RTM., and it has volume reference marks visually perceptible on the exterior of the tip. Thus, pipette tip 20' includes visual indicia 28' up to 200 .mu.L. Accordingly, the research technician can use these indicia to visually confirm the accuracy of the draw of volume adjustable pipetter 10. The volumes indicated by the reference marks 28' are only approximate volumes, and when such pipette tips are used with adjustable mechanical pipetters, these markings are intended to act only as a confirmation of the volume drawn by the mechanical pipetter.
There are numerous patents directed to pipetters, but relatively few relating to the pipette tips. In U.S. Pat. No. 4,909,991 to Oshikubo, for example, a pipetter is disclosed which is constructed in a manner that attempts to minimize the volumetric change which can occur as a result of the elevated temperature of the user's hand. In U.S. Pat. No. 4,679,446 to Sheehan et al., a pipetter is disclosed in which there are several chambers or volumes of differing size in the pipetter which can be used to draw liquid. A set of pipette tips of differing size is provided which cooperates with the pipetter to determine which fluid intake channels or pathways to the various chambers are open to draw fluid.
U.S. Pat. No. 4,237,095 to Suovaniemi is directed to a disposable pipette tip construction which is designed to produce air bubbles that do no adhere to the walls of the pipette tip during dispensing of multiple doses or volumes. Thus, when a small volume is dispensed and the pipetter piston returned to its normal position for dispensing the next volume, air bubbles are drawn into the tip and rise to the top of the liquid column remaining in the tip. The tip construction of this patent causes the air bubbles to rise in the center of the liquid column, rather than become adhered to one side or the other of the pipette tip.
Finally, U.S. Pat. Nos. 4,275,591 to Wand and 4,124,044 to Nugent both disclose protective shield assemblies which have been used with pipette tips.
As will be apparent, however, none of this patent art discloses a pipette tip construction which itself is capable of producing a draw and/or delivery of a calibrated or calibratable volume of liquid. All of these prior art references depend upon the pipetter mechanism's ability to control the drawn or delivered volume.
Traditionally, pipette assemblies have been widely used in connection with chemical and biochemical reactions. Thus, the pipette assembly is used to draw a liquid and thereafter deliver or dispense it to a container or vessel for its use in a chemical or biochemical procedure. The liquid may be reacted in the vessel with a reagent or it may be stored to permit reactions to occur over time or in response to variations in the ambient storage parameters.
This sequence of drawing and dispensing to a separate container requires in both the container and the pipette tip are contacted with the liquid, and that a liquid transfer also takes place. In some procedures, the liquid may contaminate both the pipette tip and the container as soon as it contacts the same. For other procedures, the liquid can be dangerous, making transfer to the container a safety hazard.
Existing pipette tips, however, have not been constructed in a manner allowing their use not only to draw liquid, but also as a reaction vessel in which the liquid can be stored and/or reacted with various reagents.
Accordingly, in one aspect of the present invention, it is an object to provide a pipette tip which is capable of accurately dispensing a calibrated liquid volume independently of the accuracy of the volume calibration of the pipetter.
It is another object of the present invention to provide a disposable pipette tip which facilitates pipetting accuracy.
Still another object of the present invention is to provide a pipette tip and method of pipetting which more accurately dispenses fluids despite inconsistencies in the pipetter or technician's technique.
In another aspect of the present invention, it is an object to provide a pipette tip and method which enable the pipette tip to be used not only to draw in liquids, but also to be used as a container for chemical and biochemical reactions.
It is a further object of the present invention to provide a pipette tip apparatus which is durable, compact, has a minimum number of components, is easy to use by unskilled personnel, and is economical to manufacture.
The apparatus of the present invention has other objects and features of advantage which will be more readily apparent from the following description of the Best Mode of Carrying Out the Invention and the appended claims, when taken in conjunction with the accompanying drawing.